Different techniques for the analysis of biomolecules are available on the market today. Conventional quantitative methods are still in use, as for example mass spectrometry, NMR or chromatography. A demand for more sensitive methods has resulted in development of technologies using biosensors, the most frequent are gravimetric and optical methods. However, the most sensitive way for analysing biomolecules by use of a biosensor is performed by electrochemical technology, which is based on a combination of biological molecules and electrodes.
Affinity sensors, for example immunosensors, are based on binding events between immobilized biomolecules (a ligand) and target molecules of interest (an analyte). The immobilization of the biomolecules is of vital importance for the ability to detect the binding events or interaction between the ligand and the analyte. The sensitivity obtained depends on the measuring principle of the sensor technique and the affinity properties and density of the ligand directed to the analyte.
An advantage with methods involving some affinity biosensors is that they can directly detect an interaction between an analyte in a solution and a ligand arranged at the surface of the sensor without the need of any labelled reagent, thus making the analysis less complicated and labour intense for the operator compared with competitive assays.
An affinity sensor can be used in different applications, e.g. for detecting biological contaminants, such as bacteria, viruses or toxic material thereof in tap water or in streams, or for detecting a chemical compound or a biological molecule, such as a protein or nucleic acid sequence.
In an electrochemical way, the concentration of an analyte in a solution can be calculated by measuring changes in dielectric properties, when the analyte interacts with the ligand arranged on the affinity surface of the biosensor electrode. For example, capacitive measurements or impedance measurements have been investigated for detecting different analytes.
A capacitive biosensor can be constructed by arranging capturing biomolecules (the ligand) in a thin layer on a working electrode, which previously has been coated by a thin insulating layer. The electrode is typically made of a noble metal, e.g. gold, but may also be made from other conducting materials. Then, the working electrode is arranged in a flow cell and is subjected to a potential pulse. At injection of the analyte into the flow cell, a complex of ligand-analyte is formed on the surface of the electrode due to the interaction between the analyte and the ligand, which will change the dielectric properties of the biosensor, for example the capacitance of the sensor will decrease. Hence, the analyte concentration of the solution can be evaluated by periodic measurements, before and after injection of the analyte, via measurement of the capacitance change.
WO 99/14596 describes a capacity affinity sensor based on an assembled monolayer on an electrode with immobilized recognition elements that are available to the analyte in a surrounding solution. The electrode is selective to those molecules in the solution that show affinity to the recognition elements on the surface.
A label-free immunosensor for the direct detection of cholera toxin (CT) is described in the article “Sub-attomolar detection of cholera toxin using a label-free capacitive immunosensor”, in the paper Biosensors and Bioelectronics 25 (2010) 1977-1983. In this study the concentration of CT was determined by potentiostatic capacitance measurement, i.e. by detecting the change in capacitance caused by the formation of antibody-antigen complexes. This technique is based on the electrical double-layer theory for measuring changes in dielectric properties when an antibody-antigen complex is formed on a transducer surface. The capacitance measurement was determined from the current response obtained when a potentiostatic step of +50 mV was applied to the working electrode.
However, known methods using biosensors for measurement of changes in dielectric properties present several weaknesses.
Conventional capacitive measuring devices, as disclosed in the prior art, are sensitive for external electronic disturbances, such as background noise, which will affect the variability, and thus the accuracy of the measurement.
A drawback is that the working electrode has to be exchanged for a new one, when one or more measuring series have lapsed, due to the sharp potential input commonly used. This potential input will also affect the sensitive layer of biomolecules (biorecognition layer) and the affinity of the sensor in such way that the ligand may be partly denatured. The working electrode is finally worn out and has to be exchanged, resulting in a time-consuming operation.
Hence, one disadvantage is that several of the initial capacitance measurements in a measuring series have to be used for calibrating the electrode. This calibrating operation contributes to decrease the amount of relevant unknown samples that can be run on one sensor electrode before it needs to be replaced.
A critical step in designing capacitive biosensors is the immobilization of the layer of biorecognition elements on the electrode. If it is not sufficiently insulated, ions can move through the layer, causing short-circuiting of the system, leading to a decrease in signal or absence of signal. Interferences from redox couples in the electrolyte solution can also cause high Faradic background currents, and might increase the resistance current and decrease the capacitance response.
It is desirable to have an improved method for measuring capacitive changes when using a biosensor, and a more stable system for measuring a capacitance of a biosensor to increase the sensitivity and accuracy of the method.